A protein complex (PC) suspension exhibits asymmetric biooxidation activities in the absence of any added cofactor such as NAD(P)+ or FAD. It can be extracted from pea protein (PP)-gel (PP encapsulated with Ca2+ alginate gel and aerated in air for several hours) using hot water by rotary shaking and powdered by the following three steps: (1) forming precipitates from the suspension using 30% (w/v) aqueous (NH4)2SO4, (2) crosslinking the precipitates with 0.25% (v/v) GA, and (3) preparing the cross-linked powder by freeze-drying. The cross-linked PC (CLPC) performed asymmetric oxidation of the toward (R)-isomers of rac-1 and rac-2 in 50 mM glycine-NaOH (pH 9.0) buffer/DMSO cosolvent [2.07% (v/v)] with high enantioselectivity; thus, the (S)-isomers can be obtained in greater than 99% ee from the corresponding rac-p-substituted naphthyl methyl carbinol (rac-1 and rac-2). The CLPC activity was not only competitively inhibited by addition of either 1.0 mM ZnCl2 or a chelating agent such as 1.0 mM EDTA but also denatured by pretreatments: autoclaving at 121°C (20 min) or using 6.0 M guanidine-HCl containing 50 mM DTT. These results indicated that the PC catalytic process may utilize an electron transfer system incorporating a redox cation (e.g., Fe2+ ⇄ Fe3+ or Zn). Therefore, the newly introduced CLPC can asymmetrically oxidize the substrates without the addition of any cofactor resulting in a low-cost organic method. Overall, our results show that the CLPC is an easily prepared, low-cost reagent that can function under mild conditions and afford stereoselectivity, regioselectivity, and substrate specificity.
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